An inhibitor of lymphocyte function released by crowded lymphoid cells (L-1210 leukemia) has been identified. The following studies are planned during the next 12 months: 1. Dose response studies of inhibitor production. Cells will be crowded at densities of 1 X 10 to the 8th power and supernatants will be harvested at 4, 8, 16, and 24 hours and tested against the PHA and other mitogen responses of mouse spleen cells. Inhibitor production will be correlated with cell viability at time of supernatant harvest. 2. We have already identified the fact that the inhibitor suppresses DNA synthesis of PHA-stimulated lymphocytes after 4 hours of exposure; RNA synthesis and protein synthesis will also be evaluated at short exposure intervals. 3. Since cellular proliferation in the lymphoid system is regulated by intracellular levels of adenyl and guanyl cyclase, the effect of inhibitor on cyclic nucleotide metabolism of stimulated and unstimulated mouse spleen cells, as well as lymphoid cell lines will be studied. Adenyl and guanyl cyclase activity, intracellular levels of cyclic AMP and cyclic GMP, and effects on cyclic AMP and cyclic GMP phosphodiesterase will be tested. 4. A major thrust in the coming 12 months will be refined physico-chemical studies of the inhibitor since most of the in vitro biological properties have been clarified. We will examine the following: inhibitor sensitivity to DNAase, RNAase, and pronase; purification and fractionation of inhibitor on sephadex G-200 columns; companion purification and separation of inhibitor by sucrose density gradient centrifugation; testing of fractions for biological activity from the above described procedures; polyacrylamide gel electrophoresis and isoelectric focusing characterization of inhibitor peaks with biological activity. 5. With purified inhibitor, in vivo effects on mouse immune function will be tested. These will include the primary antibody response measured in serum and by plaque-forming cell assay, development of delayed-hypersensitivity to BCG and SRBC and effects on graft rejection. 6. When the inhibitor has been purified, its site of action (or at least localization) will be determined by radioautography with radiolabeled inhibitor, and by blocking studies with antiserum made by rabbits against the inhibitor. 7. The lymphocytic chalone characteristics of the inhibit (Text Truncated -Exceeds Capacity)